PROJECT SUMMARY Despite the known roles of chromatin remodeling complexes in driving more than 20% of human cancer, the role of chromatin remodeling complexes in conferring therapeutic response in pediatric solid tumors is much less understood. Rhabdoid tumor requires residual SWI/SNF activity for transformation and progression. However, it is not known if SWI/SNF and its effect on the underlying epigenome is therapeutically targetable and if a compound targeting this complex will be successful. Furthermore, SWI/SNF has been implicated in epigenetic mechanisms of resistance suggesting this complex may be able to both confer sensitivity and resistance depending on the cancer context. Fusion positive alveolar rhabdomyosarcoma (ARMS) gains chemo-resistance without the simultaneous gain of mutations to drive this resistance. These data indicate ARMS relapse may be driven by epigenetic mechanisms. Therefore, the overall objective of this study is to define the role of chromatin structure in conferring therapeutic sensitivity in rhabdoid tumor (Aim 1) and resistance in rhabdomyosarcoma (Aim 2). I have identified mithramycin as a SWI/SNF inhibitor that induces epigenetic reprogramming and durable tumor regression in rhabdoid tumor. A consequence of mithramycin treatment is amplification of H3K27me3, a novel therapeutic vulnerability as well as the restoration of chemosensitivity. The overall goal of the F99 phase (Aim 1) is to identify synthetic lethalities that arise from SWI/SNF inhibition. Specifically, aim 1.1 will define inhibition of H3K27me3 histone demethylases KDM6A/6B as a therapeutic vulnerability in rhabdoid tumor. Aim 1.2 will define the mechanism of mithramycin-dependent chemosensitivity. These goals will build advanced expertise in mechanistic pharmacology, high-throughput sequencing, and in vivo modeling of combination therapies. In contrast to RT which is known to be chemo-refractory, alveolar rhabdomyosarcoma is initially responsive to chemotherapy before gaining resistance. Therefore, the K00 phase of this fellowship (Aim 2) will define the role of chromatin remodeling in fusion positive alveolar rhabdomyosarcoma therapeutic resistance (ARMS). Aim 2.1 will identify the chromatin remodeler that coordinates with PAX3/7-FOXO1, the oncogenic transcription factor that drives ARMS transformation and progression. Aim 2.2 will profile chromatin remodeling during the establishment of chemoresistance in an established ARMS mouse model. This phase will expand expertise in genomic approaches to include single-cell genomics and in vivo modeling to include transgenic models. In summary, this study addresses the need for a mechanistic investigation into the role of chromatin remodeling in driving therapeutic response in pediatric solid tumors. Data and training acquired in this phase will prepare me for a career exploring epigenetic mechanisms of therapeutic resistance in pediatric sarcomas.